Proteolysis of each of the complement proteins C3-C5 gives rise to aminoterminal cationic fragments with signalling molecules called anaphylatoxins (6-9). The most potent of these, C5a, elicits the broadest responses. Considering the components of the inflammatory response as margination and infiltration of leukocytes, release of granule-bound proteolytic enzymes, production of activated oxygen and nitrogen-derived radicals, changes in blood flow and capillary leakage, along with the ability to contract smooth muscle, the C5a molecule is the “complete” pro-inflammatory mediator. At sub-nanomolar to nanomolar levels, the C5a molecule elicits chemotaxis of all myeloid lineages (neutrophils, eosinophils and basophils, macrophages and monocytes), and causes vascular permeability which is markedly potentiated by prostaglandins and circulating leukocytes. Higher nanomolar concentrations elicit degranulation and activation of NADPH oxidase. This breadth of bioactivity contrasts with other inflammatory mediators. C5a has been implicated in the pathogenesis of rheumatoid arthritis, psoriasis, sepsis, reperfusion injury, and adult respiratory distress syndrome (Gerard, et al. Ann. Rev. Immunol. 12:775-808 (1994); Murdoch, et al. Blood 95(10):3032-43 (2000)).
The activities of C5a are mediated by the binding of the C5a to its receptor (C5aR). C5aR belongs to the family of seven transmembrane G-protein-coupled receptors. C5aR is a high affinity receptor for C5a, with a Kd of about InM, and is located on a number of different cell types including leukocytes. The number of receptors per cell is extremely high, up to 200,000 sites per leukocyte. Biological activation of the receptor occurs over the range that saturates binding.
The C5aR structure conforms to the seven transmembrane receptor family, with the extracellular N-terminus being followed by seven transmembrane helices connected by interhelical domains alternating as intracellular and extracellular loops, and ending with an intracellular C-terminal domain. C5aR contains an extended N-terminal extracellular domain. This large N-terminal domain is typical of G-protein coupled receptors which bind peptides including the IL-8 and fMet-Leu-Phe (FMLP) receptor families.
Inhibition of the C5a responses with C5aR antagonists should reduce the acute inflammatory response mediated via C5a without affecting other complement components. To this end, C5aR peptide antagonists and anti-C5a receptor antibodies have been previously described (Watanabe, et al. J. Immunol. Meth. 185(1):19-29 (1995); Pellas, et al. J. Immunol. 160(11):5616-21 (1998); Konteatis, et al. J. Immunol. 153(9):4200-5 (1994); Kaneko, et al. Immunol. 86(1):149-54 (1995); Morgan, et al. J. Immunol. 151(1):377.88 (1993)). For example, WO 95/00164 (Scripps Research Inst.) describes antibodies directed against an N-terminal peptide (residues 9-29) of the C5a receptor.
WO 03/062278 (G2 Therapies) describes antibodies directed to extracellular loops of C5aR other than the N-terminal domain which are effective in inhibiting C5a binding to C5aR. Specific monoclonal antibodies disclosed in this application are MAb 7F3, MAb 12D4 and MAb 6C12. Of these monoclonal antibodies, MAb 7F3 has the highest binding affinity for C5aR.